Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92198
標題: 定位PEGylation試驗提供鈉離子調控ASBTNM構型改變的證據
Site-Directed PEGylation Studies with ASBTNM Provide Evidence for Sodium-Dependent Conformational Changes
作者: 蕭瑜萱
Yu-Hsuan Hsiao
關鍵字: 膽酸轉運蛋白;二級主動運輸;交替通透機制;膜蛋白;ASBT;Secondary active transporters;Alternating access mechanism;Pegylation;Scanning mutagenesis;FSEC
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摘要: 
膽酸在肝細胞中以膽固醇為原料進行生合成,並透過膽道(biliary tract)分泌至小腸,其主要的功能是在小腸中幫助脂質和脂溶性維生素的吸收。透過腸肝循環,90%以上分泌到小腸的膽酸在協助脂質吸收之後會被再次回收到肝臟。而位於迴腸絨毛細胞膜上的膽酸運輸蛋白Apical Sodium-dependent Bile Acid Transporter (ASBT)對於膽酸回收扮演重要的角色。過去的研究認為透過抑制ASBT的膽酸回收會增加體內膽固醇的消耗,因此ASBT已經被鎖定為開發抗高血膽固醇藥物的目標之一。近期已發表來自Neisseria meningitidis之細菌同源蛋白質ASBTNM及Yersinia frederiksenii之同源蛋白ASBTYf的晶體結構共呈現三種構型,推論ASBT可能是利用不同的構型與受質結合並調控跨膜轉運,然而鈉離子調控ASBT構形改變的詳細分子機制仍然未知。本實驗利用一個能與cysteine共價結合的巨分子mPEG-Mal-5K,並結合in-gel fluorescence 來觀測ASBT-GFP螢光融合蛋白在PAGE膠體電泳上的條帶偏移,藉此探索在鈉離子梯度不存在的狀況下,該巨分子是否能進入ASBTNM的結合口袋,並評估鈉離子對蛋白質構型改變之影響。初步的結果顯示將鈉離子置換成鉀離子後,ASBTNM會形成向膜外開的構型使巨分子mPEG-Mal-5K更深入進入ASBTNM結合口袋中T112C的位置,推斷鈉離子在構型轉變上具重要的調控角色。此外利用mPEG-Mal-5K是否標記T112C所建立的實驗平台,將有助於篩選不同構型的ASBTNM點突變蛋白。

Bile acid homeostasis plays a regulatory role in controlling the level of cholesterol in the body. About 50% of cholesterol is eliminated from the body by its conversion into bile acids. Cholesterol elimination could be further increased by blockage of transporters that re-absorbs bile acids for recycling via the enterohepatic circulation. Apical Sodium-dependent Bile acid Transporter (ASBT) is the secondary active transporter for bile acid reabsorption. It utilizes the sodium gradient to drive uphill transport of bile acids across the membrane. Because of the substrate specificity ASBT has been targeted for drug development against hypercholesterolemia. The crystal structures of ASBT homolog from Neisseria meningitides (ASBTNM) and Yersinia frederiksenii (ASBTYf) were reported recently. From the structures, we proposed a putative mechanism of bile acid transport. However, it remains unclear how sodium ions trigger a conformation change from inward- to outward-facing conformation, and how substrate binding mediates an inverse conformation change to facilitate substrate uptake into the cells. We utilized the GFP-based pipeline designed for high-throughput target screening for integral membrane proteins. If the pointed mutated cysteine on the transport pathway forms a covalent link with mPEG-Mal-5K, one can observe a pegylated band using in-gel fluorescence, reflecting its accessibility for the probe. The results provide an evidence that Na+ ions mediate the conformational changes of ASBTNM and T112C is an conformational indicator for seeking mutants at alternative conformation.
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